Autism spectrum disorders (ASD) are a group of neurodevelopmental diseases that occur in as many as 1 in 150 children in the United States. Three hallmarks of autism are dysfunctional communication, impaired social interaction, and restricted and repetitive interests and activities. Even though no single genetic defect has been ascribed to having a causative role in the majority of ASD cases, twin concordance studies and rare familial forms of the disease strongly support a genetic malfunction and a combinatorial effect of genetic risk factors may contribute to the variability in the symptoms. One major obstacle to ASD research is the difficulty in obtaining human neural tissue to model the disease in vitro. Mouse models of ASD are limited since only rare genetic mutations have been identified so far, and single mutations in those genes cannot fully reproduce the range of critical behaviors characteristic of ASD. Direct reprogramming of patient tissues to induced pluripotent stem (iPS) cells and derivation of forebrain neurons from them will provide much needed insight into the molecular mechanism of neuronal dysfunction in diverse individuals on the autism spectrum. The use of patient-derived stem cells to characterize cellular defects brings together two investigative approaches. One is the identification of common cellular and molecular mechanisms that are central to deficiencies across diverse populations of patients. The other is quantitative comparison of pathological features that address differences amongst diverse patients. Our major goal is to characterize the synaptic dysfunction using concrete, quantifiable parameters in human neurons that have specific mutations in key synaptic proteins. This approach will give us a handle into the molecular synaptic complexes that may also be altered in sporadic ASD cases and could help us develop drug strategies that can normalize synaptic function. Although several groups are interested in generating iPS cells from autistic patients, these efforts generally do not have genomic information on the patients, and the large diversity of mutations associated with autism could lead to large variation in synaptic phenotypes. By focusing on generating iPS cells from patients carrying mutations in a small number of critical synaptic proteins and characterizing the molecular components of this complex, we are likely to be in a strong position to identify novel molecular defects associated with autistic synapses. Relative biochemical comparisons of wildtype and mutant protein complexes could help us find ways to restore synaptic function in ASD.

Statement of Benefit to California:

Many children in California are affected by autism spectrum disorders, which include monogenic syndromes such as Fragile X syndrome and Rett syndrome. However, the majority of cases are idiopathic and an interplay of multiple genetic risk factors is suspected. Since no current drug therapies exist for autism and an accurate diagnosis can only be made in early childhood by largely behavioral criteria, the cost of care and social burden for such a disorder is high, not to mention the devastation to the quality of life for the families of affected children. We would like to identify a core set of proteins found in synapses that are disrupted or dysregulated in autism by a biochemical approach. If we succeed in this effort, we may be able to identify novel biomarkers and molecular targets for specific patient profiles, and by cross-correlating the genetic background to specific behavioral traits in specific individuals, we may come up with molecular targets that are able to address particular symptoms, which should greatly aid in therapeutic regimens that complement existing behavioral therapies. Generating iPS neurons with known copy number variations associated with autism would be a major resource for other laboratories in California and in the field in general. The economic benefit to California is manifold, as many pharmaceutical and biotech companies in California will want to exploit these novel cell lines and the therapeutic targets identified through them in order to design better drugs for autism.

Review Summary:

Project Synopsis:
This project will study the assembly and function of nerve synapses using neurons derived from induced pluripotent stem cells (iPSCs) from autistic patients with known mutations in genes involved in synaptic function. The project comprises three specific aims. The goal of Aim 1 is to generate iPSCs and differentiate them into neurons from the dorsal and ventral regions of the forebrain. Specific Aim 2 proposes to examine synapse assembly, structure, and function. In addition, the expression of a gene linked to autism will be modified to model changes that occur in autism. Specific Aim 3 will use protein pull-down methods to identify other proteins that bind to the mutant synapse proteins and may be targets for the development of new therapeutics.
Significance and Innovation:
- Autism is a frequent disorder with a significant societal impact and whose molecular mechanism is not known.
- If successful, the results of this study are likely to be significant for understanding the cellular basis for autism and identifying novel drug targets. These studies may also provide a better understanding of synapse assembly and function.
- The investigators are using an interesting disease model and employing innovative approaches to study an important problem. The proteomic approaches of Aim 3 are creative.
Feasibility and Experimental Design:
- The proposed studies are logical extensions of both the applicant’s expertise and what is known about autism pathophysiology. The approach appears to be scientifically sound and technically feasible with substantial preliminary data.
- The rationale for selecting the genetic loci to be targeted was not clearly presented.
- Questions were raised about the feasibility of recruiting patients with copy number variations (CNV) in the loci to be targeted, which represent only 1% of the autistic population. In fact, reviewers commented that it might take more than a year to find a single patient meeting the necessary specifications.
- There was no discussion of the rationale for using postmortem tissue to study protein-protein interactions, nor the possible variability that might be introduced by this source.
- The applicant did not discuss the degree of abnormality that is expected in CNVs and whether defects would be detectable. Similarly, no discussion was provided to address how each CNV might influence expression of numerous other genes, nor how this would impact the stoichiometry within the protein complex to be studied.
- Reviewers cautioned that iPSC generation can lead to CNV, and thus it will be important to verify that the CNV under study is relevant to autism and not due to the reprogramming process.
- There was a general lack of experimental detail, particularly in Aim 3. The proposal included little in the way of alternative directions, and the list of published references was scant.
Principal Investigator (PI) and Research Team:
- Reviewers praised the PI, who is well known and respected for studying synaptic structure and function. The PI represents a great strength of the proposal and has mastered the techniques of iPSC generation.
- Strong collaborations with demonstrated clinical and proteomics expertise enhance the proposal.
Responsiveness to the RFA:
- The project fits well within the RFA, using autism patient-derived iPS cells to study possible molecular mechanisms of disease.